DE2216727B2 - Process for the production of 1-histidine by fermentation - Google Patents

Description

Preferred antibiotics are illustrated by penicillin, streptomycin, cephalosporin, cycloserine, Chloramphenicol, Erythromycin, Tetracycline Bacitracin, Kanamycin, Spiramycin, Viomycin, Novobiocin, Colistin and the like. Preferred surfactants are exemplified by anionic surfactants Agents such as alkyl sulfates, alkyl aryl sulfonates, alkyl phosphates, amide sulfonates, ester sulfonates and amide sulfates; cationic surfactants, such as amines and their salts, quaternary ammonium salts, pyridinium salts and picolinium salts; amphoteric betaine type, sulfuric acid ester type and sulfonic acid type surfactants; and non-ionic surface-active agents, such as polyoxyethylene alkyl ethers, Polyoxyethylene alkyl phenol ether, polyoxyethylene alkyl ester, Sorbitan alkyl esters, polyoxyethylene sorbitan alkyl esters, Polyoxyethylene glycol ester, polyoxyethylene glycol ether and the like.

Preferred organic and inorganic inhibitors are illustrated by mercuric chloride, Acrinol, resorcinol, cresol and the like.

It has been found that among these agents, penicillin and the nonionic surfactants Means are particularly suitable for the purpose of the invention. The one in the present description The term "penicillin" used means penicillin G, "-phenoxyethyl-penicillin, phenoxymethyl-penicillin, Amcillin, carbenicillin and various other synthetic penicillins of the known Types.

The conditions under which the growth-inhibiting Means used according to the invention are very important and in biochemical terms too very interesting.

In the case that a growth retardant which has strong activity is used, it is advantageous that the additive used at a later stage of the logarithmic growth Tribal is made. In particular, the addition gives an earlier stage of logarithmic growth in the case of using a 1-histidine producing strain which is derived from a 1-glutamic acid producing strain has been obtained by mutation treatment - as shown in the examples -, an increase in the production of a relatively large amount of glutamic acid while adding in a larger amount at a later stage in the logarithmic growth of the stem used of 1-histidine without the production of 1-glutamic acid brings forth.

The amount of the growth inhibitor having a strong activity added is advantageously between 1 and 100 y / ml, preferably between 1 and 50 y / ml of the medium. On the other hand, nonionic surfactants which have a lower inhibitory effect at an earlier stage of the logarithmic growth of the stem used are added, preferably in an amount of 100 to 2000 y / ml of the medium. The 1-histidine accumulated in the breeding broth can be recovered after conventional method, e.g. B. with the help of chromatography or an ion exchange resin.

Table 1 below shows those obtained according to the invention Results with and without the addition of the growth-inhibiting agent, the K salt of penicillin G. The technical advantage of the present invention is clearly demonstrated.

Table 1

K-SaIz
Penicillir
Addition time of
is G
Encore
lot Dry-
cellular
weight 1-histidine Amount of
produ
ornamental
1-histidine
Cell
substance (Hours) (y / ml) (mg / ml) (mg / ml) (mg / mg) 15th 5 40.9 7.14 0.175 18th 10 37.4 10.62 0.284 No addition 50.1 5.70 0.114

Comment on paragraph 1:

tribe

The 1-histidine producing strain Corynebacterium glutamicum ATCC 21 604 was used. The seed culture was formed as described in Example 1 below.

Composition of the medium

20% sucrose, 0.2% KH ₂ PO ₄ , 0.1% K ₂ HPO ₄ , 0.05% MgSO ₄ • 7H ₂ 0.0.001% FeSO ₄ • 7H ₂ 0.0.001% MnSO ₄ • 4H ₂ 0 , 0.5% corn steep liquor, 200 γ / l biotin, 2.0% (NHi) ₂ SO ₄ and 0.2% urea.

Main culture

The main culture was carried out in a manner similar to that described in Example 2 below.

As can be seen from the results shown above, increases by the addition of a growth inhibitor Substance for the culture broth the formation and accumulation of the 1-histidine significant.

The following examples illustrate the invention.

Example 1 I. Strain

The 1-histidine producing strain Corynebacterium glutamicum ÄTCC 21604 was used.

II. Seed culture

20 ml of a medium consisting of: 4% glucose, 2% peptone, 1% meat extract, 0.5% yeast extract, 0.05% MgSO ₄ 7H ₂ O, 0.15% KH ₂ PO ₄ , 0.05% K ₂ HP-0.05% MgSO ₄ -7H ₂ O, 0.15% KH ₂ PO ₄ , 0.05% K ₂ HPO ₄ , 30y / l biotin and 0.3% urea. The seed culture, adjusted to pH 7.2, was placed in a 250 ml Erlenmeyer flask and sterilized by heating. The strain was inoculated on the medium and cultured ^{at 28 ° C. with shaking for 24 hours.}

III. Main culture

40 ml of a medium consisting of: 20% sucrose, 0.2% KH ₂ PO ₄ , 0.1% K ₂ HPO ₄ , 0.05% MgSO ₄ -7H ₂ 0.0.001% FeSO ₄ · 7H ₂ 0, 0.001% MnSO ₄ - 4H ₂ O,

0.5% corn steep liquor, 200 γ / 1 biotin, 2% (NH _{4 ) 2} SO 4 and 0.2% urea adjusted to pH 7.2 were placed in a Sakaguchi flask and sterilized by heating ; then it was inoculated with 2 ml of the seed culture. After cultivation with shaking at 28 ° C. for 24 hours, various antibiotics and organic and inorganic inhibitors in various concentrations, which were prepared separately under sterile conditions, were added to the respective fractions of the cultivation broth as shown in Table 2. Each fraction was further grown with shaking for 48 hours.

The amounts of! -Histidine that was accumulated in each broth when different inhibitors are shown in Table 2.

Table 2

Additives additive
lot Dry-
cellular
weight 1-histidine (ml) (mg / ml) (mg / ml) K-SaIz des Penicillins G .... 2.5 17.4 6.94 K-SaIz des Penicillins G .... 5 16.3 7.08 K-SaIz des Penicillins G. 10 15.2 7.98 Antibiotics Cycloserine 10 18.7 7.01 20th 15.9 6.82 Cephalosporin ... 10 16.8 7.11 20th 15.7 6.63 Streptomycin .... 5 16.2 5.38 10 15.1 5.55 Inorganic Inhibitor Mercury chloride .. 2 14.0 4.90 More organic Inhibitor Acrinol 2 14.2 5.00 No addition -M. ·) M
21.1 4.55

was prepared, each of the fractions of the main culture in different stages of breeding - as in table 3 shown - added. Each fraction was then under the same conditions as described above, Cultivated for 48 to 72 hours with stirring and aeration.

The amounts of 1-histidine and! -Glutamic acid thus accumulated in the broth are shown in Table 3 shown.

Table 3

Addition to 1
of Penicil
Addition time C-SaIz
into G
Encore
lot Dry-
cellular
weight 1-histidine 1-embers
amic acid ¹⁵ (hours) (mg / ml) (mg / ml) (mg / ml) 9 10 20.2 4.6 27.5 15th 10 24.3 6.2 23.2 20th 10 42.1 9.3 0.5 ²⁰ No addition 52.2 6.0 0

The production of 1-glutamic acid does not belong the subject of the invention.

² SB eis ρ ie 1 3

The same treatment as described in Example 1 was carried out with the difference that Brevibacterium flavum ATCC 21 605 was used as a seed and various surfactants have been found Agents as shown in Table 4 were added to each fractions of the culture medium for the purpose of inhibition.

The amounts of 1-histidine thus produced in these fractions are shown in Table 4.

Example 2 I. Strain

The same strain as in Example 1 was used.

II. Seed culture

300 ml of the same medium - as described in Example 1 - were filled into a conical 2 l flask and sterilized by heating. The strain was inoculated on the medium and then cultured ^{at 28 ° C. with shaking for 24 hours.}

III. Main culture

Table 4 Surface active Encore Dry-
cellular 1-histidine Fabrics lot weight 40 (y / ml) (mg / ml) (mg / ml) Polyoxyethylene alkylamine 100 13.4 5.2 cationic 45 Alkyldimethyl benzyl ammo nium chloride ... 100 11.9 5.7 Alkyl trimethyl ammonium halo- 50 genius 100 12.1 5.1 anionic Sulfonic acid salt of oleic acid amids 10 10.5 5.0 55 amphoteric Alkyl betaine 100 11.5 5.3 non-ionic Sorbitan trioleate .. 1000 21.0 7.1 No addition 22.1 4.1 60

Three of the same medium as described in Example 1 were placed in a 5-1 vibrating fermenter and sterilized by heating. 300 ml of the seed culture were inoculated, and were grown with agitation (650 rpm) and aeration at 28 ⁰ C and at a pH of 6.8. Thereafter, 10 y / ml K salts of penicillin G, which were separated under sterile conditions

Example 4

The same treatment as described in Example 1 was carried out, but with the difference that both the various 1-histidine-producing strains listed in Table 5 and 10 y / ml des Penicillin G K salt were used.

The amounts of 1-histidine thus accumulated are shown in Table 5.

Table 5

1-histidine without addition with addition from Trunk used from penicillin penicillin (mg / ml) (mg / ml) Corynebacterium glutamicum ATCC 4.60 21607 7.13 ■) ^vu Brevibacterium flavum 4.51 ATCC 21 605 6.61 Arthrobacter citreus 1.53 ATCC 21 600 2.41 Bacillus megaterium 2.05 ATCC 21 603 3.20

As shown in the above examples, the addition of growth inhibitors improves the yield essential to 1-histidine.

Claims (8)

1 2 significant, like ζ. B. as an additive to medicaments, patent claims · foodstuffs, feedstuffs and the like. The present invention relates to a method for the production of 1-histidine by aerobic fermentation 1. Method for the production of 1-histidine 5 using a microorganism which is imdurch Aerobic fermentation using stalls is to produce 1-histidine, being the micro one Microorganism capable of growing 1-histidine organism in a culture medium which produce, the microorganism in ches carbon sources, nitrogen sources and anorgaeinem Culture medium is grown which contains niche salts, which is characterized Sources of carbon, sources of nitrogen and inorganic is that the culture medium is a growth inhibitor Contains salts, thereby adding a characteristic agent. This makes it possible to draw 1-histi, that one din to the culture medium in high concentration and in good yield added growth inhibitor. produce and accumulate so that the 1-histidine 2. The method according to claim 1, characterized can be obtained industrially economically, draws that the cultivation takes 48 to 96 hours 15 The present invention is based on the discovery a temperature of 25 to 35 ° C and with a kung that it is possible to produce 1-histidine in better yield pH 5 to 8 is carried out. and to produce at higher concentration, and 3. The method according to claim 1, characterized by the addition of a growth-inhibiting agent draws that the growth retardant to the culture medium to the growth of the inside of 24 hours from the start of the cultivation of the microorganism. Besides, the processing is added to the medium. Addition of the growth-inhibiting agent 4. The method as claimed in claim 3, characterized in that it is advantageous because it is possible to that the growth inhibitor from cleaning by other undesirable microorganisms Group selected from antibiotics to prevent men. Surface-active agents, organic and an- 25 The use of a growth inhibitor organic inhibitors. Mean is already used in the production of 1-glutamine 5. The method according to claim 4, characterized in that the acid has been applied by fermentation, however draws that the growth inhibitor is in the art in the case of the fermentation of is an antibiotic that has never been reported from the group-1-histidine. Chooses from Penicillin, Streptomycin, 30 The present invention is described in the following Cephalosporin, cycloserine, chloramphenicol, Ery- description to be further described, thromycin, tetracycline, bacitracin, kanamycin, a seed culture of a microorganism that Spiramycin, Viomycin, Novobiocin, and Colistin is what is supposed to produce 1-histidine on one consists. , Main culture medium inoculated, which suitable human 6. The method according to claim 4, characterized in that it contains carbon sources, nitrogen sources, organic sources, that the growth inhibitor is a niche salting and - if desired - another is a surfactant that is selected from the group containing nutrients and under aerobic conditions is selected from alkyl sulfates, alkylaryl at a temperature of 25 to 35 ° C and at a sulfonates, alkyl phosphates, amide sulfonates, pH from 5 to 8 grown in no more than 24 hours. Ester sulfonates, amide sulfates, amines and their 4 ° Then the medium is a suitable amount of one Salts, quaternary ammonium salts, pyridine growth inhibitors added, and the salts, picoline salts, amphoteric surface-active cultivation is carried out under the same conditions of the ven agents of the betaine type, of the sulfuric acid-first breeding continued with the difference that ester type and sulfonic acid type, polyoxy- the cultivation time is 48 to 72 hours, so that in ethylene alkyl ether, polyoxyethylene - alkylphenol- 45 the medium a large amount of 1-histidine product ether, Polyoxyethylene alkyl ester, sorbitan alkyl zated and accumulated. ester, polyoxyethylene sorbitan alkyl ester, polyoxy- The 1-histidine producing microorganisms, ethylene glycol esters and polyoxyethylene glycol ethers which exist for the purpose of the present invention, can be turned, are illustrated by the 7. The method according to claim 4, characterized in that 50, which belongs to the genera Brevibacterium, Coryne, that the growth inhibitor bacterium, Arthrobacter, Micrococcus, Bacillus and is an organic or inorganic agent owned by Nocardia, although any and all of them is selected from the group consisting of mercury strains capable of producing 1-hisitidine, un-'chloride, There is acrinol, resorcinol and cresol. harms their taxonomic classification 8. The method according to claim 1, characterized in that 55 can be used. Such microorganisms draws that the growth inhibitor in are good in the art for the production of 1-histidine the medium in an amount of no more than known (e.g. German Offenlegungsschrift 2 031 387). 2000 gamma / 1 is available. All mentioned in the description below Strains have been deposited in the American Type Culture Collection ⁶⁰ and are freely available to the public. Growth retardants used for the purpose the present invention can be used, basically include any and all substances, ⁶ 5 that have antibiotic properties, such as antibiotics, and both surface-active substances as well as The commercial production of 1-histidine is various organic and inorganic inhibitory pathways Its various important uses substances.